Identification of Linear B Cell Epitopes on CD2V Protein of African Swine Fever Virus by Monoclonal Antibodies

ABSTRACT The CD2-like (CD2V) protein is a crucial antigen of African swine fever virus (ASFV). CD2V interacts with the cellular AP-1 protein, participates in intracellular transport of virus, and induces neutralizing antibodies to partly protect swine from virus attack. In this study, a specific CD2V dimeric protein was designed to enhance antigenicity and immunogenicity, expressed in a Bac-to-Bac baculovirus expression vector system and purified by Ni-affinity chromatography. After animal immunization, five monoclonal antibodies (mAbs) (7E12, 22B3, 18A3, 13G11, and 43C2) against CD2V were developed. The variable regions of heavy chains and light chains of the mAbs were sequenced to prove that the five mAbs differed from one another. The mAbs of CD2V could combine with ASFV by immunoperoxidase monolayer assay (IPMA). B cell epitopes of CD2V were screened using the five mAbs by indirect enzyme-linked immunosorbent assay (ELISA) and Dot-ELISA. Therefore, three B cell epitopes (147FVKYT151, 157EYNWN161, and 195SSNY198) were identified. This is the first time that mAbs of the ASFV CD2V protein have been developed and the sequencing of heavy chains and light chains of mAbs has been completed. Linear B cell epitopes, which were core targets of immunoprotection of the CD2V protein, were identified by mAbs for the first time. This study provides efficient epitopes for the development of ASFV subunit vaccines. IMPORTANCE The ASFV CD2V protein is a crucial antigen on the outer envelopes of virus particles. A modified ASFV CD2V dimeric protein was expressed in the Bac-to-Bac baculovirus expression vector system. Five monoclonal antibodies (mAbs) against CD2V were developed, sequenced, and applied to identify CD2V protein B cell epitopes. Three B cell epitopes, 147FVKYT151, 157EYNWN161, and 195SSNY198, were identified. This is the first time CD2V mAbs have been developed, the sequencing of heavy chains and light chains of CD2V mAbs have been completed, and CD2V B cell epitopes have been identified by using scanning peptide method and bioinformatics methods.


Analysis of immunogenicity for CD2V recombinant protein
Healthy female BALB/c mice, which were about 20g and 6-8 weeks old, were selected as immune experimental animals. The experimental group and the control group were set with five mice in each group. 5 µg/mouse CD2V recombinant protein was as immunogen in the experimental group, and the immunogen of the control group was sterilized PBS. Freund's complete adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for following two immunizations. Three immunizations were carried out in total. And the interval of immunization was 3 weeks (21 days). Serum was got by cutting tail at 63 days. The titer of antibody to CD2V recombinant protein in serum was detected using indirect ELISA method.

Amplifying variable regions of heavy chain and light chain of monoclonal antibodies
RNA of cells producing the five monoclonal antibodies was extracted with TRIzol (Invitrogen, Thermofisher,US) and reverse-transcribed into cDNA by PrimeScript TM RT Master Mix (Takara, Dalian, China). Nucleic acids of the heavy and light chains of CD2V mAbs were amplified by PCR using the primers in Table S2, and the results were observed by 2% agarose gel electrophoresis.

Prediction of CD2V B cell epitopes
Continuous B cell epitope regions of ASFV CD2V proteins were predicted using four bioinformatics softwares--ABCPred, BCPREDS, BepiPred and SVMTriP.
Continuous B cell epitope regions of ASFV CD2V proteins were determined by comparison of the predicted results of each software.

Bioinformatics analysis and modification of ASFV CD2V protein
As was shown in Fig. S1 A and B, 1-206 amino acids (aa) was extracellular area including 1-16 aa CD2V signal peptide sequence. 207-229 aa was across the membrane area, and 230-360 aa was intracellular area. This result was consistent with the topology of ASFV CD2V in the Uniprot protein database (Fig. S1C). According to ProtParam online prediction software, the isoelectric point (PI) of CD2V full-length sequence was 6.21 and the molecular weight was 41.008kDa, while the PI of CD2V extracellular region (1-206aa) was 4.65 and the molecular weight was 23.68kDa.
CD2V recombinant protein was designed using a flexible linker (GGGGSGGGGSGGGGS) coupling with two homologous CD2V extracellular sequences in figure S1D. The PI of CD2V recombinant protein was 5.36 and the molecular weight was 45.62kDa by ProtParam tool of Expasy.

The codon optimization of CD2V-dimer sequence
The result of codon optimization for the original amino acid sequence of CD2V was shown in Figure S2. The codon optimization increased the CAI value to 0.94 ( Fig.   S2B) and the average GC content to 49.34% ( Fig. S2C) in order that CD2V could be expressed well in eukaryotic baculovirus expression system.

Construction of a recombinant vector pFastBac TM 1-CD2V and development of Bacmids
As was shown in Figure S3A, 1% agarose gel electrophoresis showed that sequence of the CD2V recombinant protein was 1362 bp. As was shown in Figure S3B, two positive clones, pFastBac TM 1-CD2V-1 and pFastBac TM 1-CD2V-6, were located on correct size (3662 bp). The sequences of pFastBac TM 1-CD2V-1 and pFastBac TM 1-CD2V-6 were consistent with the CD2V target gene sequence. PCR results of 14 clones from blue-white selection were all positive clones. pFastBac TM 1-CD2V(DH10Bac)-1 was selected to prepare Bacmid.

Immunogenicity analysis of ASFV CD2V recombinant protein
As was shown in Figure S4, the antibody titer of CD2V recombinant protein as immunogen was 1:12,800 after third immunization. The OD450 values of negative control were lower than 0.5.

Amplification of variable region of heavy and light chain in monoclonal antibodies
As was shown in Figure S5, the sizes of the heavy and light chain were approximately 300 bp. And the heavy chain was slightly larger than the light chain. The CDR regions of mAbs were verified by IMGT. These analyses of IMGT were shown in Figure S6.

Bioinformatics prediction of ASFV CD2V protein B cell epitopes
As was shown in Figure S7A, ABCPred predicted continuous B cell epitopes on the extracellular region of the CD2V protein as the score from high to low: As was shown in Figure S7B,  As was shown in Figure S6D,